Purification of host factor required for in Vitro transcription of poliovirus RNA

VIROLOGY

128.245-251

Purification

(1983)

of Host

Factor

Required

for in Vitro Transcription

of Poliovirus

RNA

ASIM DASGUPTA Department

of Microbiology & Immuno~ UCLA School of Medicine, Received

February

and Jonsson Cwnqwehensive Cancer Los Angeles, California 9002.4

10, 1983; accepted

March

Center,

25, 1983

A host cell protein (host factor) which is required for the in vitro transcription of poliovirus RNA has been purified to near homogeneity from an uninfected HeLa cell ribosomal salt wash. A single protein with an approximate molecular weight of 67,009 is associated with this “host factor” activity. The purified host factor catalyzes the synthesis of genome-length copies of poliovirion RNA in the presence of poliovirus RNA polymerase. Oligo(U) can replace host factor in this reaction. The RNA product synthesized in the presence of host factor is shown to be complementary to virion RNA.

The single-stranded RNA genome of poliovirus is replicated by an RNA-dependent RNA polymerase (replicase) found in the cytoplasm of cells infected with poliovirus (I). The detailed mechanism involved in the replication of poliovirus RNA genome is not fully understood. The first step in the replication of viral RNA should be the synthesis of a complete negative-strand RNA molecule (2). Previous studies have shown that both the virus-specific poly(U)polymerase and a host factor are required for the in vitro transcription of poliovirion RNA (3). Highly purified poliovirus polymerase (NCVP4, P63) (4) has been shown to catalyze the synthesis of complete negative-strand RNA molecules in the presence of an oligo(U) primer (5). Recently, a host protein with an approximate molecular weight of 67,000 has been purified from the cytoplasm of uninfected HeLa cells and shown to catalyze the synthesis of genome-length copies of poliovirion RNA in the presence of viral polymerase (6,7’). Antibody to partially purified host factor has been shown to inhibit host faetor-dependent synthesis of negative-strand RNAs (8). A host factor has also been implicated in the replication of encephalomyocarditis virus (9). In this communication, we describe purification of host factor from a ribosomal salt wash fraction prepared from uninfected HeLa cells and analyze 245

the composition of host factor using SDSpolyacrylamide gel electrophoresis (SDSPAGE). We also demonstrate that the synthesis of genome-length copies of poliovirion RNA is catalyzed by purified host factor and viral polymerase. The in vitro synthesized RNA is shown to be complementary to virion RNA. The infection of HeLa cells with poliovirus and the purification of replicase through phosphocellulose (fraction II) and poly(U)-Sepharose 4B (fraction IV) were as described (3, 10). Poliovirion RNA was prepared by the method described by Spector and Baltimore (II). Figure 1 shows poly(U)-polymerase and host factor-dependent repliease activities of fractions eluted from a poly(U)-Sepharose column (50-400 mMKC1 gradient) and the SDS-PAGE pattern of the corresponding fractions containing [s%Jrnethionine-labeled viral proteins. Active polymerase contained mainly NCVP4 (P63), NCVP2, and a noncapsid protein (32,000) which comigrated with poliovirus capsid protein VP2. Traces of three other viral proteins (47K, 49K, and NCVPlb) were also found to correspond to replicase activity. Although these viral proteins copurified with the polymerase, the intensities of these proteins varied from one batch of polymerase to the other. Fresh preparations always contained more 0042-6822/83 Copyright All rights

$3.00

Q 1983 by Academic Press, Inc. of reproduction in any form reserved.

246

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7

9

II

COMMUNICATIONS

13 15

FRACTION NUMBER

FIG. 1. Analysis of viral proteins present in fractions (from a poly(U)-Sepharose 4B column) having poliovirus-specific poly(U)-polymerase activity. (Left) Poliovirus proteins were labeled with [%]methionine (NEN, 1070 Ci/mmol, 2 mCi per 4 X 10’ cells). Cells were then lysed and purification of poly(U) polymerase through phosphocellulose and poly(U)-Sepharose 4B columns was carried out (9). Fractions from the poly(U)-sepharose 4B column were assayed for poliovirus-specific poly(A) . oligo(U)-dependent poly(U)-polymerase activity at 30” for 30 min as described by Flanegan and Baltimore (18) (0). The same fractions were also assayed for poliovirion RNA-dependent replicase activity in the absence (A) and in the presence (A) of purified host factor. The standard incubation mixture contained, in a total volume of 50 91: 50 mMHEPES (pH 8.0), 5 mMMg-acetate, 4 mM dithiothreitol, 10 pg/ml actinomycin D, 0.2 m&f each of three other ribonucleoside triphosphates, 1 9&f [(Y-q]CTP (sp act 20,000~50,000 cpm/pmol), and where indicated 0.03 9g of fraction VI host factor (Table 1). Incubation was for 1 hr at 30”. Labeled products were collected on membrane filters and counted. (Right) Corresponding fractions containing [%]methionine-labeled viral proteins were analyzed on a 10% SDS-polyacrylamide gel (19) alongside [asS]methionine-labeled poliovirus-infected extract (extreme right lane). The gel was fixed with 10% acetic acid and fluorographed, and then an autoradiogram was prepared. Molecular weights of different polypeptides were calculated on the basis of the relative migration of standard protein markers analyzed on the same gel. The autoradiogram was purposely overexposed to visualize the 49K and 47K bands. In some preparations of [%]methionine-labeled poly(U)-polymerase, the 63K band (NCVPI) appeared as a doublet.

NCVPlb than the other two. Whereas aged preparations invariably contained more of 49K and 4i’K proteins and very little NCVPlb as shown in Fig. 1. It is interesting to note that antibody to the poliovirus 5’-terminal protein VPg has recently been shown to immunoprecipitate two viral precursor polypeptides to VPg which are very similar to the 49K and 47K proteins described here (1.2, 13). In addition, a monoclonal antibody, that precipitates

NCVP4, also reacts with the 32K protein, suggesting that this protein is derived from NCVP4 (to be published elsewhere). Host factor activity was originally detected in the high salt wash (0.5 M KCl) fraction of ribosomes prepared from uninfected HeLa cells (3). We, therefore, developed a host factor purification scheme starting from HeLa ribosomal salt wash. While this manuscript was in preparation, Baron and Baltimore reported that most

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a phosphocellulose column (1 X 10 cm) previously equilibrated with buffer B. Proteins were then eluted from the column by an 80-ml, 50-600 mM linear KC1 gradient in the same buffer. Peak fractions (eluted around 350 mM KCl) were pooled and dialyzed against buffer C (10 mM potassium phosphate, pH 7.8,5 mM mercaptoethanol, 1 mM EDTA, and 10% glycerol) (fraction IV). Fraction IV host factor was then applied to a hydroxyapatite column (1 X 5 cm) previously equilibrated with buffer C. Proteins were eluted by a 40-ml, lo-300 mM linear gradient of potassium phosphate. The host factor activity was eluted as a single peak around 60-70 mM phosphate. Active fractions were pooled, concentrated against solid sucrose, and dialyzed overnight against buffer B (fraction V). Fraction V host factor was further purified by lo-30% glycerol gradient (5 ml in buffer B) centrifugation at 45,000 rpm for 18 hr using a Beckman SW 50.1 rotor.

of the host factor (SO-SO%) was located in the soluble cytoplasm of HeLa cells (6). Previously, our inability to detect host factor in the cellular cytoplasm was due to high background caused by Sloe fractions during the host factor assay. Table 1 summarizes the purification of host factor from uninfected HeLa cells. Preparation of ribosomal salt wash from 2 X 10” HeLa cells was as described (fraction I) (3). A 40-60% saturated ammonium sulfate cut of fraction I host factor was prepared. Precipitated proteins were collected by centrifugation, dissolved in buffer A (20 mM TrisHCl, pH 8.0,150 mM KCl, 1 mM EDTA, 5 mM mercaptoethanol, and 10% glycerol), and dialyzed overnight against buffer A (fraction II). Fraction II host factor was then applied to a DEAE-Sephacel column (2 X 20 cm) previously equilibrated with buffer A. Host factor did not bind to the column at 150 mM KCl. The column was washed with buffer A and the pooled proteins from flow-through and wash fractions were concentrated by O-80% ammonium sulfate precipitation. Precipitated proteins were taken up in 20 ml buffer B (buffer A containing 50 mM KCI) and dialyzed overnight against the same buffer. The dialysate (fraction III) was applied to

Host

Fraction I. II. III. IV. V. VI. VII.

Ribosomal salt wash 40-60s Ammonium sulfate DEAE-Sephacel Phosphocellulose Hydroxyapatite Glycerol gradient Second phosphocellulose

Note. Uninfected HeLa carried out as described ’ One unit of host factor of 1 pmol of labeled CMP (3) at 30” for 1 hr under host factor were assayed

OF HOST FACTOR

factor

activity

sedimented

as a 4 S

peak (fraction VI). Active fractions were pooled and, after adjustment of the KC1 concentration to 150 mM, applied to a second phosphocellulose column (0.5 X 4 cm). Proteins were eluted by a 20-ml, 150-600 mM linear KC1 gradient in buffer A. Ac-

TABLE PURIFICATION

247

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1

FROM UNINFECTED

Total protein (mg)

Total activity (units)”

100 45 15 6 1 0.20 0.03

30,000 35,000 32,000 27,000 18,000 15,000 9,000

cells (2 X 10“‘) were collected by in the text. activity is defined as the amount into acid-precipitable form in the standard RNA synthesis assay immediately after elution from

HeLa

Specific activity (units/mg)

CELLS

Purification (fold)

Yield (%I)

1 2.6 7.1 15 60 250 1000

100 116 106 90 60 50 30

300 777 2,133 4,500 18,000 75,000 300,000 centrifugation

and purification

of host

factor

was

of protein required to catalyze the incorporation presence of 1 Gg of fraction IV poly(U)-polymerase conditions (3, 8, 10). All the fractions containing the columns,

248

SHORT

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tive fractions were pooled, concentrated against solid sucrose, and dialyzed overnight against buffer B (fraction VII). The purification scheme described here yielded host factor which was purified lOOO-fold with a specific activity of 300,000 units/mg protein as defined (Table 1). Fraction VII host factor can be kept frozen at -70” for several months without detectable loss of activity. Figure 2 shows an SDS-PAGE pattern of two different preparations of purified host factor (fraction VII, Table 1). A single protein with an approximate molecular weight of 67,000 was associated with host factor activity. In some preparations of purified host factor, another slightly slower moving protein of approximate molecular weight 74,000 was observed. However, when individual fractions from the phosphocellulose column were assayed for host factor-dependent replicase activity, only the 67K protein was found to correspond exactly to host factor activity (data not shown). Baron and Baltimore recently purified host factor from the cytoplasm of uninfected HeLa cells (6). A single polypeptide of apparent molecular weight 67,000 was associated with host factor activity. Thus, by the criterion of molecular weight, it appears that the host factor activities found in the cytoplasmic and ribosomal fractions are most probably represented by the same polypeptide. However, this point needs further study. To examine the RNA products catalyzed by the viral polymerase-host factor combination, we analyzed the [a-32P]CMP-labeled RNAs synthesized in response to virion RNA on a denaturing agarose gel (14). Synthesis of labeled RNA products including 35 S RNA molecules was observed in the reactions containing both purified host factor and viral poly(U)-polymerase (Fig. 3, lanes 3 and 4). Synthesis of fulllength RNA also occurred when oligo(U) was substituted for the host factor (lanes 5 and 6). As expected, in the absence of either polymerase (lanes 7 and 8) or host factor (lane 2), no RNA synthesis could be detected. It is clear then that in the presence of either fraction VII host factor or

JI-

87-

45-

30-

FIG. 2. Analysis of purified host factor by SDSpolyacrylamide gel electrophoresis. Host factor was purified from two different batches (batch 1,2 X 10”’ cells; batch 2, 1 X 10” cells) of uninfected HeLa cells according to the procedure described in the text. Fractions (20 ~1) from freshly prepared host factor preparations (fraction VII, Table 1) were analyzed (batch 1 in lane 1 and batch 2 in lane 2) on a 10% SDS-polyacrylamide gel according to Laemmli (19). Proteins were visualized by Coomassie blue staining. The following molecular weight markers were also analyzed on the same gel: phosphorylase b (94,000); bovine serum albumin (67,000); ovalbumin (45,000); and carbonic anhydrase (30,000). Batch 1 and 2 host factor preparations had specific activities of 300,000 and 320,000 units/mg protein, respectively.

oligo(U), the viral poly(U)-polymerase is capable of synthesizing full-length RNA. As reported earlier (.3), the purified host factor (fraction VII)-polymerase combination copied only poly(A)-containing RNAs and showed a preference for

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FIG. 3. Analysis of in vitro synthesized RNA on a denaturing agarose gel. One microgram of poliovirus replicase (fraction IV) (3) was incubated in the absence (lane 2) and in the presence of 0.01 (lane 3) and 0.02 (lane 4) pg of fraction VII, batch 2 (Fig. 2, lane 2) host factor with 1 fig poliovirion RNA as described in the legend to Fig. 1. Eight and sixteen picomoles of oligo(U) were used (instead of host factor) in the reactions analyzed in lanes 5 and 6, respectively. Lanes 7 and 8 were the same as lanes 3 and 4, respectively, except that poliovirus replicase was omitted from these reactions. In lane 1, a sample of =P-labeled poliovirion RNA was analyzed. Incubation was for 1 hr at 30”. [(u-32P]CTP was used as the labeled ribonucleoside triphosphate. Labeled products, after phenol extraction, were precipitated from the aqueous phase in the presence of 5 pg carrier yeast tRNA. Precipitated RNAs were collected by centrifugation, washed twice with ethanol, lyophilized, and dissolved in buffer containing 15 mM methylmercuric hydroxide and were then analyzed on a 1% agarose gel eontaining 15 mM methylmercuric hydroxide as de-

249

copying poliovirion RNA compared to other poly(A)-containing RNAs (data not shown), The time course for the purified host factor-dependent replicase reaction did not change significantly compared to the result published earlier (3). Full-length RNA (35 S) was detected in 30 min in the presence of 5 miV MgZ+ (data not shown). No detectable RNA synthesis was observed when the complete reaction mixture was depleted of either virion RNA or M$+. All four ribonucleoside triphosphates were required for the synthesis of 35 S RNA molecules (data not shown). To examine the products of the replicase reaction for complementarity to template, we determined its sensitivity to RNase digestion before and after hybridization with excess unlabeled viral RNA (Table 2). About 90% of the labeled RNA synthesized in response to viral RNA was resistant to RNase digestion, indicating that most of the product RNA was in a doublestranded form. When the product RNA was denatured, about 80% of it became sensitive to RNase digestion. When denatured RNA was reannealed without any added RNA, 35% of the labeled RNA was found to be RNase resistant. This could be due to the partial renaturation of the labeled product with the large molar excess of unlabeled template present in the reaction. In order to demonstrate that the product RNA was of the negative polarity, the denatured product was hybridized to excess virion RNA or HeLa cytoplasmic RNA. Only virion RNA could drive the hybridization reaction to completion so that all of the product RNA was resistant to RNase digestion. The majority of the product RNA, therefore, appears to be complementary to virion RNA. Using a highly purified preparation of

scribed by Bailey and Davidson (24). For unknown reasons, the labeled band in lane 5 moved slightly faster than the 35 S RNA marker. The dye front in lane 5 also moved faster compared to those in other lanes. However, when an aliquot of the same sample was reanalyzed, the band was found to comigrate with the 35 S RNA marker.

250

SHORT TABLE

RIBONUCLEASE BEFORE

RESISTANCE AND AFTER EXCESS

2 OF THE PRODUCT ANNEALING WITH

POLIOVIRUS

None Boiled and quickly chilled Boiled and annealed with no RNA Boiled and annealed with HeLa RNA Boiled and annealed with polio RNA

RNA

RNA

RNase Treatment

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resistance (%I 92 20 35 35 100

Note. 32P-Labeled product RNA was precipitated from a standard reaction with cold 5% TCA. The precipitate was dissolved in 10 mMTris-HCl (pH 7.5) and reprecipitated with ethanol. The precipitate was dissolved in 10 mM Tris-HCI (pH 7.5) and divided into five aliquots. Each aliquot had 30,000 cpm (Cerenkov). Samples were then boiled for 5 min and annealed for 60 min at 51’ after addition of 0.2 M NaCl (final concentration) in the presence or absence of added RNA (8 pg HeLa or polio). Samples were then chilled and divided into two aliquots. One aliquot from each sample was treated with RNases Ti, Ta, and A for 30 min at 30’. Samples were then precipitated with 5% TCA, collected on membrane filters, and counted.

poly(U)-polymerase (4), Van Dyke et aL have characterized the oligo(U)-primed, in vitro synthesized product RNA by conducting a nearest-neighbor analysis and by comparing the resulting distribution of dinucleotides in the minus-strand RNA (5). Their results suggested that true heteropolymeric copies of the template RNA were synthesized by the poliovirus RNA polymerase in vitro. We have used both criteria in further characterizing the host factorstimulated replicase products. Our results were very similar to those reported by Van Dyke et al (5) (data not shown). The best studied viral replicase is that of Q/3 bacteriophage. The Q@replicase consists of one phage protein (the polymerase) and three host proteins, which are all required for the initiation of RNA synthesis (15). A fourth cellular protein, al-

though not a subunit of the replicase complex per se, is also involved in the initiation of phage RNA synthesis (16, 17). To date, the poliovirus replicase activity has been shown to be dependent on only one host protein (host factor) ((3, 6) and this report). It is logical to presume that the initiation of RNA synthesis for the eukaryotic “plus-strand” RNA viruses is a complex process and may require the participation of more than one host protein. Although we have used an apparently homogeneous preparation of host factor, we cannot preclude the possibility of involvement of other host cell proteins in poliovirus replication. In fact, we have observed other cellular proteins in our polymerase preparations used in this study (unpublished observation). The purification scheme described by Van Dyke and Flanegan (4) yields a nearly homogeneous preparation of poly(U)-polymerase which is essentially free of contaminating host cell proteins. Future studies using such purified preparations should allow us to further examine the role(s) of additional host proteins in poliovirus replication. ACKNOWLEDGMENTS This work was supported by Grant AI 18272 from the National Institute of Allergy and Infectious Diseases. The author wishes to thank Dr. David Baltimore for sending copies of Refs. (6, 7, 12) before their publications. The author gratefully acknowledges the excellent secretarial help of Laurie Patierno during the preparation of this manuscript. The author wishes to thank Dr. Casey Morrow for correcting the manuscript. REFERENCES 1. BALTIMORE,

D., EGGERS,

H.

J., FRANKLIN,

R. M.,

and TAMM, I., Proc. Nat. Acad Sci USA 49, 843449 (1963). 2. BALTIMORE, D., J. MoL BioL 32,359-368 (1966). 3. DASGUPTA, A., ZABEL, P., and BALTIMORE, D., CeU 19,423-429 (1980). 4. VAN DYKE, T. A., and FLANEGAN, J. B., J. vird 35.732-740 (1930). 5. VAN

DYKE, T. A., RICKLES, R. J., and FLANEGAN, J. B., J. BioL Chm 257,4610-4617 (1982). 6. BARON, M. H., and BALTIMORE, D., J. Bid Chxm. 257,12351-12358 (1982).

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7. BARON, M. H., and BALTIMORE, D., J. Biol Chem 257,12359-12366 (1982). 8. DASGUPTA, A., HOLLINGSHEAD, P., and BALTIMORE, D., J. viral. 42, 1114-111’7 (1982). 9. DIMITRIEVA, T. M., SHEHEGLOVA, M. V., and AGOL, V., Virology 92, 271-277 (1979). 10. DASGUPTA, A., BARON, M. H., and BALTIMORE, D., Proc. Nat. Acad. Sci. USA 76,2679-2683 (1979). 11. SPECTOR, D. H., and BALTIMORE, D., J. Viral. 15, 14181431 (1975). 1.2. BARON, M. H., and BALTIMORE, D., Cell 28, 395404 (1982). 13. SEMLER, B. L., ANDERSON, C. W., HANECAK, R., DORNER, L. F., and WIMMER, E., Cell 28, 405412 (1982).

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14. BAILEY, J. M., and DAVIDSON, N., Anal B&hem. 70,75-85 (1976). 15. KAMEN, R. I., in “RNA Phages” (N. Zinder, ed.), pp. 203-234. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1975. 16. FRANZE DE FERNANDEZ, M. T., EOYANG, L., and AUGUST, J. T., Nature (London) 219, 588-590 (1968). 17. SHAPIRO, L., FRANZE DE FERNANDEZ, M. T., and AUGUST, J. T., Nature (London) 220, 478-480 (1968). 18. FLANEGAN, J. B., and BALTIMORE, D., Proc. Nat. Acad Sci. USA 74, 3677-3680 (1977). 19. LAEMMLI, U. K., Nature (London) 227, 680-685 (1970).